Project Summary Pseudomonas aeruginosa (Pa) is a major human pathogen whose virulence is predicated on its ability to form biofilms - slimy layers of polysaccharides and bacteria that confer resistance to antibiotics and immune clearance. Pa biofilms are particularly problematic in Cystic Fibrosis (CF), where they are a major cause of mortality. We have uncovered novel roles for Pf bacteriophages in chronic biofilm infections. We recently reported that Pf phages produced by Pa organize host and microbial polymers present in sputum into a liquid crystal. This crystalline architecture makes CF sputum more viscous and more adhesive. It also traps antibiotics like tobramycin and prevents them from reaching the bacteria living within. Along with these effects on biofilm formation and function, Pf phages directly interfere with host immunity. Purified Pf phage inhibits phagocytosis and hampers the ability to clear bacterial infections. In preliminary work with a cohort of Pa infected CF patients, we found associations between Pf phage and Pa lung infection burden, chronicity of Pa infection, declines in pulmonary function during exacerbation, and antibiotic resistance to anti- Pseudomonal antibiotics. Given the high abundance of Pf in CF sputum we also postulate that Pf liquid crystals may affect host mucociliary clearance by attachment and impairment of the epithelial ciliary brush function. In light of these exciting preliminary data, we hypothesize that Pf phage cause worse clinical outcomes in CF by disrupting bacterial clearance mechanisms and promoting bacterial tolerance to antibiotics. We will test this hypothesis in experiments with the following aims: In Aim 1 we will determine how Pf phage impacts cell function in the airways. Our hypothesis is that Pf phage interferes with phagocytosis, impairs mucus transport and disrupts the function of the ciliary brush. To test this, we will conduct a number of in vitro experiments to investigate the mechanisms of Pf pathogenecity. In Aim 2 we will elucidate how Pf phage production drives Pa antibiotic tolerance. Our hypothesis is that the liquid crystalline organization of Pa biofilms prevents diffusion of antibiotics and promotes the emergence of resistant strains. To test this, we will assess how Pf phage-mediated sequestration impacts the bioactivity of antibiotics and the emergence of antibiotic resistant Pa isolates over time. In Aim 3 we will characterize how Pf phage impacts clinical outcomes in CF patients. Our hypothesis is that higher Pf phage titers are associated with crystalline sputum and worse clinical outcomes. To test this, we will perform longitudinal studies to determine how Pa colonization influences pulmonary function and the occurrence of exacerbations. We will also investigate in CF patients for the presence of antibody responses directed at Pf and correlate their presence with clinical outcomes. This proposal represents a bold and radically unconventional approach to Pa biofilm infections and CF pathobiology. If successful, this work will identify a novel therapeutic target in CF and other settings where Pa biofilm infections cause disease.